The general goal of this project is to learn how to design and engineer metalloenzymes with heteronuclear metal assemblies such as the heme-manganese site in manganese peroxidase (MnP), the heme-copper site in heme-copper oxidases (HCOs), and the heme-iron site in nitric oxide reductase (NOR). Specifically, we would like to elucidate the structure/function relationship in these proteins. MnP is a heme peroxidase involved in biodegradation of lignin and bioremediation of aromatic pollutants. HCOs are a superfamily of terminal oxidases in cellular respiratory chains that include cytochrome c oxidase. The lack of HCOs or the presence of their mutant forms is linked to Alzheimer's disease, Leigh syndrome, and aging. NOR catalyzes the two-electron reduction of NO to N2O. It is one of the key enzymes in the inorganic nitrogen cycle responsible to returning dinitrogen to the atmosphere. The study of NOR may provide structural and spectroscopic model for mammalian enzymes that produce and utilize NO in signal transduction pathways. Stable, easy-to-produce, and well-characterized heme proteins (such as cytochrome c peroxidase (CcP) and myoglobin (Mb)) will be used as scaffolds for making biomimetic models of MnP, HCO and NOR. The focus will be on relating the structures of the metal-binding sites to their functions. More importantly, these model systems will be used to demonstrate how metal-binding site structure can be fine-tuned to realize different functional properties. Site-directed mutagenesis and expressed protein ligation will be used to introduce natural and unnatural amino acids, respectively, into the metal-binding sites. This will probe the role of the metal ligands and residues in the secondary coordination sphere on the structure and function of the metal-binding centers.